Oxidative stress is one of the major contributing factors in alcohol-mediated cell and tissue damage. The majority of reactive oxygen and nitrogen species (ROS/RNS) by alcohol is being produced through direct inhibition of the mitochondrial respiratory chain and induction of ethanol-inducible cytochrome P450 2E1 (CYP2E1) and other enzymes such as inducible nitric oxide synthase. Despite the well-established roles of ROS/RNS in alcohol-induced cell injury, the proteins that are selectively oxidized by ROS/RNS are poorly characterized. By using biotin-N-maleimide (biotin-NM) as a specific probe, we developed a sensitive method to positively identify oxidized cysteine residues in alcohol-exposed hepatoma cells. During this fiscal year, we have applied this targeted proteomics approach to identify oxidized proteins in alcohol-exposed mouse liver. The oxidized proteins, purified with streptavidin-agarose beads, were resolved by 2-D gel electrophoresis. Protein spots, that displayed differential abundances in alcohol-fed mouse livers compared to those in the pair-fed controls, were excised from the 2-D gels, in-gel digested with trypsin and subjected to mass spectrometry. The mass spectrometric analysis of more than 90 proteins revealed that many proteins involved in chaperone activities (heat shock proteins, protein disulfide isomerase, etc.), anti-oxidant defense (catalase, peroxiredoxin, thioredoxin, etc.), intermediary metabolism including the transmethylation pathway (methionine S-adenosyl transferase, S-adenosylhomocysteine hydroxylase, etc.) and cytoskeletal proteins (actin, keratins, etc) were oxidized in alcohol-fed mouse livers. Our results may explain the underlying mechanisms for the inactivation of some of these enzymes leading to the reduced levels of antioxidants such as S-adenosylmethionine with the increased levels of homocysteine observed in alcohol-exposed animal tissues and alcoholic human subjects as reported by many scientists. Rapid inactivation of peroxiredoxin through sulfinic/sulfonic acid formation of its active site cysteine was demonstrated within 1 h after ethanol exposure in E47 HepG2 cells (with transduced CYP2E1). Similar inactivation of peroxiredoxin through sulfinic/sulfonic acid formation was also observed in alcohol-exposed mouse liver but not in pair-fed control animals. Oxidative inactivation of peroxiredoxin and catalase may contribute to the elevated levels of peroxides observed in E47 HepG2 cells and alcohol-exposed animal tissues. We believe that inactivation or functional loss of some of these oxidized proteins may contribute to increased sensitivity toward ethanol-mediated oxidative injury. From our proteomics studies, we observed that mitochondrial aldehyde dehydrogenase (ALDH2) was oxidized or S-nitrosylated after ethanol-sensitive E47 HepG2 cells were exposed to ethanol. To study the functional implication of oxidative modifications of ALDH2, we studied the effects of various NO donors on potential inactivation of ALDH2 via S-nitrosylation. Our results showed that NO donors such as S-nitrosoglutathione (GSNO) and S-nitroso-N-acetyl-D,L,-penicillamine (SNAP) reduced the ALDH2 activity by S-nitrosylation and that the suppressed ALDH2 activity could be restored by addition of a cell permeable glutathione analog GSH-ethylester (GSH-EE). To directly verify S-nitrosylation of ALDH2, we immunopurified the mitochondrial ALDH2 proteins from untreated hepatoma cells, cells treated with GSNO alone, or cells treated with GSNO plus GSH-EE. Under the condition where one immunopurified ALDH2 protein was identified for all samples, S-nitrosylated protein, detected by anti-S-nitrosylated Cys antibody, was observed only from the cells treated with GSNO alone but not in two other samples. These data strongly suggest that the active site cysteine (Cys302) of ALDH2 was reversibly S-nitrosylated by NO donors. Similar observation of S-nitrosylated ALDH2 was observed in alcohol-fed rats but not in pair-fed control rats, suggesting that S-nitrosylation of ALDH2 can take place in in vivo conditions. Our results may also explain the underlying mechanism for the reduced levels of ALDH2 activity in alcoholic individuals and after exposure to toxic chemicals, which could increase the levels of NO. We have recently reported selective and persistent activation of c-Jun N-terminal protein kinase (JNK) by many substrates of CYP2E1 such as acetaminophen (APAP), 4-hydroxynonenal, carbon tetrachloride, and long chain fatty acids. We also observed that ethanol and non-CYP2E1 substrates such as troglitazone and staurosporine (STS) activated JNK and p38 protein kinase (p38 kinase). Our results also showed that ethanol, acetaminophen and STS caused translocation of proapoptotic Bax to mitochondria prior to apoptosis in a time-dependent manner. Activation of both JNK and p38 kinase seemed important in STS- or ethanol-induced mitochondrial translocation of activated Bax and cell death, because pretreatment with a respective inhibitor of JNK or p38 kinase significantly reduced the activity of each kinase and the rates of mitochondrial translocation of Bax and apoptosis. In addition, by immunoprecipitation followed by 2-D gel analysis and protein sequencing, we have identified beta5-tubulin as a new Bax interacting protein which retains Bax in resting states. Other proteins such as Ku70, heat shock protein 60 and 14-3-3, that were recently reported to retain Bax in the cytoplasm of un-stimulated cells, were not immunoprecipitated along with Bax protein when HepG2 cell exrtracts were immunoprecipitated with the specific anti-Bax antibody or anti-beta-tubulin antibody. These results suggest strong interaction between Bax and beta-tubulin but not with other proteins aforementioned. Our results also showed that various cell death stimulants such as staurosporine, etoposide, and hydrogen peroxide activated JNK and p38 kinase, which directly phosphorylated Bax before it was translocated to mitochondria to initiate apoptosis. JNK- and p38 kinase-mediated phosphorylation Bax itself led to its activation (exposure of its N-terminus detected by the conformation specific anti-Bax monoclonal antibody 6A7) and its release from its beta-tubulin in HepG2 hepatoma cells. Phosphorylation of Bax was demonstrated by the shift of the pI value of Bax (from 5.1 to 4.0) on 2-D gels and confirmed by metabolic labeling with 32P-inorganic phosphate. Reconstitution experiments showed that beta-tubulin but not actin, used as a negative control, sufficiently retained the recombinant Bax protein and that JNK or p38 kinase directly phosphorylated Bax before the phosphorylated Bax was released from beta-tubulin. Taken together, our results suggest that JNK- and p38 kinase-mediated phosphorylation of Bax leads to its activation, which might disrupt the previous interaction between the N-terminal domain and the C-terminal transmembrane domain of Bax. Exposure of the C-terminal transmembrane domain is likely to lead to mitochondrial translocation of activated Bax to initiate apoptosis process. Our results represent new data about the role of beta-tubulin as a new Bax-retaining protein and a new mechanism by which activated Bax is released from beta-tubulin through JNK- and p38 kinase-mediated phosphorylation. Our results may explain the underlying mechanisms for the positive relationship between activation of JNK or p38 kinase and apoptosis caused by many distinct cell death stimulants or conditions, as reported by many scientists.